Collagens, the most abundant proteins in connective tissues of higher eukaryotes, play important structural roles in many tissues and organs. Collagen production is under strict developmental control, and each cell type produces only a subset of the total repertoire of collagens. This cell- type specificity of collagen synthesis determines, to a large extent, the architecture and physical properties of tissues. One of the best characterized examples of developmentally-regulated collagen production is the differentiation of mesenchyme into hyaline cartilage. The major collagen in prechondrogenic mesenchyme is type I; as the mesenchymal cells differentiate into chondrocytes, they stop producing type I collagen and initiate synthesis of cartilage-specific collagens, although type I collagen synthesis is reinitiated in hypertrophic chondrocytes at the chondro-osseous junction. In addition, there is evidence for abnormal reinitiation of type I collagen gene expression in osteoarthritic articular cartilage. There is relatively little information regarding the molecular mechanisms that mediate these transitions in normal and abnormal collagen production, despite their importance for skeletal development. An unusual mechanism has been discovered that prevents synthesis of the alpha2(I) collagen subunit in chick cartilage. The previously identified (upstream) promoter of the alpha2(I) collagen gene is repressed in cartilage, but an internal promoter is activated, resulting in production of an alternative transcript that does not encode alpha2(I) collagen. This change in promoter utilization effectively prevents alpha2(I) collagen synthesis in cartilage and may also result in production of a previously unknown noncollagenous protein. This renewal application proposes to: 1) continue to define the cis elements and transcription factors that mediate chondrocyte-specific activation of the internal promoter of the chick alpha2(I) collagen gene. Four domains that are involved in chondrocyte-specific transcriptional activity have been identified; the cis elements in these domains will be characterized and the transcription factors that interact with them will be identified; 2) define the cis elements and transcription factors the mediate chondrocyte-specific repression of the upstream promoter of the alpha2(I) collagen gene; 3) determine the effects of abrogated production of the alternative transcript and ectopic expression of the predicted protein; and 4) purify and characterize the small RNA-binding protein that appears to be the major product of the alternative transcript of the alpha2(I) collagen gene. This multifaceted experimental approach is intended to further our understanding of the molecular mechanisms underlying the developmentally regulated production of alpha2(I) collagen during chondrogenesis, an important process for normal skeletal development, as well as the abnormal expression in osteoarthritis.